Basic and applied histochemistry最新文献

The use of the supravital mitochondrial-specific dye Rhodamine 123 (Rh 123) in combination with flow cytometry permits the monitoring of the changes in the mitochondrial transmembrane potential, reflecting the overall mitochondrial activity of the living cell. While this probe appears to be a potent tool for these studies, it also exhibits an important limit in the interpretation of the results: it cannot distinguish between an increase in mitochondrial activity without biogenesis and a modification of mitochondrial content. 10-n-Nonyl Acridine Orange chloride (NAO) constitutes another mitochondrial specific fluorochrome. In contrast with Rh 123, NAO accumulation in the cell does not seem to be driven by the proton-motrice force but does seem to be related to specific interactions with mitochondrial membrane proteins and/or lipids. In this work, the cytotoxicity of NAO, the kinetics of cellular uptake and the release of the dye have been determined using flow cytometry. The use of several ionophores or mitochondrial inhibitors has confirmed the independence of NAO uptake regarding mitochondrial transmembrane potential. NAO was also used to examine the changes in the mitochondrial compartment during the transfer of articular chondrocytes from cartilage to the culture conditions, where Rh 123 evidenced changes in mitochondrial activity and/or biogenesis, in order to know whether the use of probes with different specificity allows one to distinguish between mitochondrial activity and biogenesis.

The activity of cytochrome c oxidase was studied in aging brain on non-synaptic and intra-synaptic mitochondria from frontal cerebral cortex, hippocampus and striatum of 4, 8, 12, 16, 20 and 24 month-old Sprague-Dawley rats. Specific activities of cytochrome oxidase were significantly higher in light synaptic mitochondria than in non-synaptic or heavy ones at all the ages examined. However, enzyme activity in light mitochondria from cerebral cortex remains unchanged during aging, being increased in hippocampus and striatum. These results indicate that aging affected not only the various cerebral area (macroheterogeneity), but also the different mitochondrial populations (subcellular heterogeneity).

The effect of a single administration of lead nitrate on the activity of gamma-glutamyltranspeptidase (gamma-GT), adenosine triphosphatase (ATPase), the placental form of glutathione S-transferase (GST-P) and adenylate cyclase (AC), four enzymes widely used as phenotypic markers for preneoplasia, was investigated in the liver of male Wistar rats. The results of the histochemical enzymatic staining indicated that an acute treatment with lead nitrate induces the activity of gamma-GT, mainly in the hepatocytes located around zone I of the liver acinus, with a maximum seen between 72-96 hours. On the other hand, the activity of ATPase was found to be severely inhibited at 2-3 days after treatment, as shown by a strong decrease in the staining of the bile canaliculi of zones II and III. Immunohistochemical analysis revealed that lead nitrate administration also resulted in the appearance in most of the hepatocytes of GST-P, an enzyme whose activity is almost undetectable in normal rat liver, but is elevated in preneoplastic liver lesions. Finally, lead nitrate treatment resulted in an inhibition of AC activity which was maximal after 24 hours.

Features of hepatic and enteric tissue have been detected in serial sections of 2 out of 6 endodermal sinus tumors (EST) and embryonal carcinomas of the testis showing endodermal sinus areas. Hepatic nests, which appeared as tubules, cords and giant multinucleated cells, were PAS-positive as well as immunoreactive for alphafetoprotein (AFP) and cytokeratin (CK). Enteric tubules were found within EST areas and were characterized by their lack of cell atypism. The presence of diffuse PAS-, AFP- and CK-reactivity in cytoplasm, together with a brush border or an apical staining for CEA, were quite distinctive. EST showed a potential for both intra- and extraembryonal endodermic development and may be interpreted as a monophyletic teratoma.

The localization of alkaline phosphatase (E.C. 3.1.3.1.) positivity during prenatal development of the hypothalamus of the rat is described. At E12 all layers of the prosencephalon display alkaline phosphatase (AP) positivity. The AP positivity increases from dorsal to ventral. Within the hypothalamic area a second, rostro-ventral gradient exists from E14 onwards. At E18 both gradients have decreased. At E20 almost all AP positivity has disappeared from the hypothalamus, with the exception of some reaction product in the dorsal ventricular matrix of the hypothalamus. The significance of this pattern in relation to the differentiation of the hypothalamus and to the formation of hypothalamic connections is discussed. It is suggested that AP activity is related to the formation of connections.

The distribution of chromogranin A and neuron specific enolase (NSE) in the neuroendocrine gut system and the morphology and distribution of cells containing gastrin, somatostatin, neurotensin and VIP in the gastroenteropacreatic (GEP) apparatus of Erinaceus europaeus were investigated by immunohistochemical methods. Chromogranin A and somatostatin immunoreactive cells were present throughout the gastrointestinal mucosa, with the exception of the oesophagus and in the pancreas. Gastrin cells were peculiar of the pyloric glands and duodenal mucosa and neurotensin cells of the small intestine. No VIP immunoreactive endocrine cells were noticed in the GEP system. VIP and NSE immunoreactivities were detected both in nerve cell bodies and terminals of the wall of the GEP apparatus. NSE immunoreactivity was found in the endocrine cells of the fundic and pyloric mucosa.

Microtubule organizing centers (MTOC) in control, irradiated and heated C3H 10T1/2 mouse embryo cells and two radiation-transformed sublines, R1 and R25, were made visible by indirect immunofluorescence using antibody against tubulin. The MTOC were reformed by 5-min incubation in fresh medium after the microtubules were depolymerized with nocodazole. The R1 line had a different distribution of MTOC/cell than the parent 10T1/2 line or R25, which had similar distributions. After irradiation, multiple MTOC appeared in the normal and radiation-transformed cells irradiated to 10 Gy and incubated for 24 or 48 h. The multiple foci of microtubule reformation in the irradiated cells indicate that radiation damage is expressed in structural elements in the cytoplasm. After heat treatment of the three cell lines (43 degrees C for 93 min and 45 degrees C for 25 min), the MTOC were disrupted and many cells did not have visible organizing centers at 24 or 48 h, while others had a large number of small centers of microtubule reformation. The distribution of MTOC/cell seen in R25 cells after the treatment had similar patterns to those of the 10T1/2 line rather than to those of the other radiation-transformed line, R1. Thus, the radiation or heat response seen in the MTOC is not dependent upon cell transformation.

Biochemical and histochemical analyses were carried out on the carbohydrate components of hare (Lepus europaeus) oviduct in anoestrous condition. Biochemical tests demonstrated that all the glycosidic components typical of glycoproteins and glycosaminoglycans are present in the ampulla and in the isthmus regions of oviduct, and that statistically significant differences exist in the sugar content between these two regions. In addition, lectin histochemistry combined with glycosidase digestion and selective histochemical stainings provided a series of rather detailed information on the localization of different neutral sugars and aminosugars. Hypotheses are advanced on the probable meaning of the different composition of ampullary and isthmic glycoconjugates in relation to the physiologically differentiated roles of the two oviduct tracts.

In non-specific and rheumatoid synovitis, the use of specific monoclonal antibodies against antigenic determinants of cells of the immune system showed that the characteristic changes of rheumatoid synovitis are located in the synovial internal layers. The monocytes were OKM1, OKM5, S100, OKDR positive, while the subintimal monocytes in non-specific synovitis were OKDR negative. We suggest that, in rheumatoid synovitis, the previously activated monocytes are transported by the bloodstream and pass through the so-called "sinovial barrier" to arrive in the subintimal layers ready to interact with T helper lymphocytes and initiate the immune response mechanisms responsible for lesions in rheumatoid synovitis.

The distribution in the periaqueductal gray matter (PAG) of substance P (SP) and Met-enkephalin (Met-enk), two neuropeptides related to pain modulation, was investigated. The study was carried out at different mesencephalic levels, with immunocytochemical methods in both "normal" and colchicine treated rats. The SP immunoreactive structures are made up of uniformly distributed fibers and neurons. The latter are particularly numerous in the ventral PAG and their number increases in a caudo-cranial direction. Along the midbrain axis, however, no significant variation of the Met-enk elements was observed. The Met-enk fibers are most numerous in lateral PAG, together with the few positive cells, which are also present. The comparison between the present results and others reported in the literature suggests that there may be species differences in the distribution of the two neuropeptides. The distribution of immunostructures has been discussed in the light of the physiological and cytoarchitectural features of PAG.